Abstract

Pemphigus vulgaris (PV) is considered as a model for an autoantibody-mediated organ-specific autoimmune disorder. IgG autoantibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3), the major autoantigen in PV, cause loss of epidermal keratinocyte adhesion, resulting in blisters and erosions of the skin and mucous membranes. The association of human autoimmune diseases with distinct HLA alleles is a well-known phenomenon, such as the association with HLA-DRB1*04:02 in PV. However, direct evidence that HLA-DRB1*04:02–restricted autoreactive CD4+ T cells recognizing immunodominant epitopes of Dsg3 initiate the production of Dsg3-reactive IgG autoantibodies is still missing. In this study, we show in a humanized HLA-DRB1*04:02–transgenic mouse model that HLA-DRB1*04:02–restricted T cell recognition of human Dsg3 epitopes leads to the induction of pathogenic IgG Abs that induce loss of epidermal adhesion, a hallmark in the immune pathogenesis of PV. Activation of Dsg3-reactive CD4+ T cells by distinct human Dsg3 peptides that bind to HLA-DRβ1*04:02 is tightly regulated by the HLA-DRB1*04:02 allele and leads, via CD40-CD40L–dependent T cell–B cell interaction, to the production of IgG Abs that recognize both N- and COOH-terminal epitopes of the human Dsg3 ectodomain. These findings demonstrate key cellular and humoral immune events in the autoimmune cascade of PV in a humanized HLA-transgenic mouse model. We show that CD4+ T cells recognizing immunodominant Dsg3 epitopes in the context of the PV-associated HLA-DRB1*04:02 induce the secretion of Dsg3-specific IgG in vivo. Finally, these results identify Dsg3-reactive CD4+ T cells as potential therapeutic targets in the future.

Introduction

Pemphigus vulgaris (PV) is a potentially lethal autoimmune bullous disease of the skin caused by IgG autoantibodies against desmoglein (Dsg)3 and Dsg1, components of the desmosomal adhesion complex, leading to loss of adhesion of epidermal keratinocytes (1). Clinically, PV presents with chronic and progressive mucosal lesions and, during the course of disease, with extensive flaccid skin blisters. Pemphigus can be considered as a paradigm of an autoantibody-mediated organ-specific autoimmune disease because the targeted autoantigens are known and its immune pathogenesis is relatively well characterized (2, 3). Although rare, PV is more common in distinct ethnic groups such as Jews, Iranians, Irakis, and Indians where the disease-associated HLA class II alleles HLA-DRB1*04:02 and HLA-DQB1*05:03 are more common (4, 5).

We and others have provided evidence that these PV-associated HLA class II alleles are involved in the activation of Dsg3-specific autoaggressive CD4+ T cells, which are critical for the induction and maintenance of autoreactive memory B cells as precursors of autoantibody-producing plasma cells. Autoaggressive CD4+ Th cell responses against the ectodomain of Dsg3 were identified in PV patients by several independent investigators (6–8). Dsg3-specific autoaggressive Th2 cells were preferentially detected in PV patients with active disease, whereas healthy carriers of the PV-associated HLA class II alleles HLA-DRB1*04:02 and HLA-DQB1*05:03 showed autoreactive Th1 cell responses against Dsg3 (8). On the basis of a peptide-binding algorithm for HLA-DRB1*04:02, Wucherpfennig et al. (6) identified several Dsg3 peptides as potential T cell epitopes. We subsequently found that distinct Dsg3 peptides, which all share a positively charged arginine (R) or lysine (K) at position 4, induce a proliferative in vitro response of peripheral T cells from PV patients. Moreover, CD4+ T cell recognition of these Dsg3 epitopes was restricted by HLA-DRB1*04:02 and, in some cases, by HLA-DQB1*05:03, which share similar peptide binding motifs and thus provides a molecular basis for the dual HLA class II association observed in PV (9).

Despite the strong evidence that IgG autoAb specific for Dsg3 or Dsg1 are pathogenic in PV, the immunological mechanisms regulating IgG autoAb formation are largely unknown. In this study, we show in an HLA-DRB1*04:02–transgenic mouse model that induction of pathogenic, Dsg3-reactive IgG Abs requires activation of Dsg3-reactive CD4+ T cells. Moreover, T cell activation critically depends on the recognition of epitopes of the Dsg3 ectodomain, which specifically bind to the PV-associated HLA class II allele, DRB1*04:02 (6, 10). Thus T cell–dependent induction of pathogenic IgG Abs in pemphigus is tightly regulated by polymorphisms of peptide-binding motifs of distinct HLA class II alleles, which are associated with PV.

Materials and Methods

HLA-DR4–transgenic mice

HLA-DRA1*01:01-DRB1*04:02/-DQA1*03:01, -DQB1*03:02 (DQ8)–transgenic DBA/1J mice were generated as described previously (11, 12). The mice also express the human CD4 coreceptor (13) and are I-Aβ deficient (I-Aβ−/−) (Supplemental Fig. 1A) (14). Transgenic C57BL/10 mice coexpressing DRB1*04:01 and human CD4, whereas lacking endogenous murine MHC class II and a functional Ncf1 gene were also used (15–17). All animal experiments were reviewed approved by the local Laboratory Animal Ethics Committees at the Philipps University Marburg and the Karolinska Institutet Stockholm, respectively. The experiments were done in compliance with local policies and guidelines on the use of laboratory animals.

Dsg3 recombinants and peptides

Human and mouse Dsg3 recombinants linked to an E tag and a 6× histidine tag were produced and purified by affinity chromatography as described previously (Table I) (8, 18–20). Fifteen- and 17-mer Dsg3 peptides were synthesized by F-moc chemistry resulting in a >95% purity (Table III) (peptides & elephants, Potsdam, Germany) and were solubilized in DMSO acetic acid buffer (stock concentration, 2 mg/ml).

Immunization of HLA-DR4–transgenic mice

Eight to 12 wk old HLA-DRB1*04:02–transgenic mice were immunized by i.p. injection of recombinant human Dsg3 (20–40 μg) in alum on day 0, followed by immunization with 20–40 μg Dsg3 in IFA (Sigma-Aldrich, St. Louis, MO) on days 14 and 28 (Supplemental Fig. 1B). Blood samples were taken to analyze anti-Dsg3 IgG by ELISA as described previously (20). For the induction of Dsg3-reactive T cell responses, mice were injected s.c. into the foot paws with human Dsg3 protein and peptides (20–40μg), respectively, in adjuvant (IFA or TiterMax) (Sigma-Aldrich) on day 0, and draining popliteal and inguinal lymph nodes were harvested on days 7–10 (Supplemental Fig. 1C).

In vitro proliferative assays

CD4+ T cells were isolated from splenocytes of Dsg3-immunized HLA-DRB1*04:02–transgenic mice by MACS (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s manual. Purity of the isolated CD4+ T cells was >90% as confirmed by FACS analysis. T cells were stained with CFSE (Molecular Probes/Life Technologies, Carlsbad, CA) following an established protocol (22). Specifically, 2 × 105 CD4+ T cells were cocultured with 104 BMDC in 96-well round-bottom microtiter plates. Proliferation of CFSE-labeled CD4+ T cells was determined by FACS analysis after 72 h of culture. In addition, IL-4+ and IFN-γ+ T cells from popliteal and inguinal lymph nodes were restimulated in vitro with Dsg3 protein and Dsg3 peptides, respectively, and were subjected to ELISPOT analysis using an ELISPOT reader (A.EL.VIS, Hannover, Germany).

Ab treatment

HLA-DRB1*04:02–transgenic mice received rat anti-mouse CD4 mAb (GK1.5) or rat IgG2b isotype control (LTF-2) (both BioXCell, West Lebanon, NH) at 200 μg i.p. on days −1, 0, 1, 3, and 5; mice were immunized with 20–40 μg Dsg3 i.p. on day 0. Mice were also injected with hamster anti-mouse CD40L mAb (MR-1) or poly hamster Ig isotype control (both BioXCell) at 500 μg i.p. on days −2, 0, 2, 4, 7, and 14 and were immunized with 20–40 μg Dsg3 i.p. on day 0.

Keratinocyte dissociation assay

The dispase based keratinocyte dissociation assay was performed as described previously (23, 24). Briefly, primary human epidermal keratinocytes were grown to confluence in CnT-57 medium in 12-well plates (CELLnTEC Advanced Cell Systems, Bern, Switzerland), switched to CnT-02 medium (CELLnTEC Advanced Cell Systems) supplemented with 1.2 mM CaCl2 24 h prior to the assay, and were incubated with mouse sera (diluted at 1:50) or the anti-Dsg3 mAb AK23 (1 μg/ml; MBL, Nagoya, Japan) overnight at 37°C. For the final 2 h of the assay, recombinant exfoliative toxin A (0.5 μg/ml; Toxin Technology, Sarasota, FL) was added. The adherent keratinocytes were then incubated with dispase I (1.5 U/ml; Roche Applied Sciences, Mannheim, Germany) at 37°C for 20 min and subjected to mechanical stress. Fragments were fixed in 1 ml of a 10% formalin solution and stained with crystal violet.

Ex vivo model using human skin biopsies

Four-millimeter punch biopsies were obtained from unaffected skin samples of patients undergoing dermatosurgery in the Department of Dermatology and Allergology at the Philipps University Marburg, Germany. The patients gave written informed consent to donate excess skin samples resulting from surgical procedures for this research project. Punch biopsies were incubated in 200 μl keratinocyte medium CnT-57 (CELLnTEC Advanced Cell Systems) supplemented with 1.2 mM CaCl2 in 96-well round-bottom plates. Serum samples of Dsg3-immunized HLA-DRB1*04:02–transgenic mice (serum samples of PBS-injected mice served as controls) were injected into the dermal site of the biospies at a dilution of 1:20–1:25. The skin biopsies were incubated overnight at 5% CO2 and 37°C, then rinsed in PBS several times, fixed in 10% formalin, and then subjected to H&E staining for histological analysis. For detection of tissue-bound IgG Abs, cryosections of the skin biopsies were subjected to immunofluorescence staining.

Immunofluorescence and histopathology

Human skin specimens and buccal or palatinal mouse mucosa were embedded in OCT TissueTek compound (Sakura, Tokyo, Japan), frozen, cut into 3- to 4-μm sections and stained using a 1:250 dilution of a rabbit anti-mouse IgG FITC-labeled Ab for direct immunofluorescence microscopy (Zymed, San Francisco, CA). Indirect immunofluorescence with serum samples of immunized mice (1:25 dilution) was performed on monkey esophagus, according to the manufacturer’s protocol (The Binding Site, Birmingham, U.K.), and modified by using a rabbit anti-mouse IgG FITC-labeled Ab (Zymed). Formalin-fixed skin and mucosa sections of Dsg3-immunized mice were also stained with H&E.

Dsg3 ELISA

Mouse sera were diluted 1:20 and analyzed for anti-Dsg3 IgG by ELISA as described previously (20). In addition, two commercial human Dsg3 ELISA kits were also used (MBL, Nagoya, Japan; Euroimmun, Lübeck, Germany) and were modified by using an anti-mouse IgG HRP-conjugated secondary Ab.

Results

Immunization of HLA-DRB1*04:02-transgenic mice with human Dsg3 leads to the induction of Dsg3-specific, pathogenic IgG Abs

We generated a humanized mouse model aimed at reproducing the immunological key findings of PV under the immunogenetic restriction by HLA-DRB1*04:02. DBA/J1 mice transgenic for HLA-DRB1*04:02 and HLA-DQB1*03:02 (which is in a linkage disequilibrium with DRB1*04:02) and the human CD4 coreceptor, which were devoid of functional murine MHC class II (I-Aβ−/−) were generated (Supplemental Fig. 1A). Mice were immunized with human recombinant Dsg3 (aa 1–566) (Table I, Supplemental Fig. 1B) and mounted a robust IgG response against human Dsg3 (Fig. 1A); these Abs belonged preferentially to the IgG1 and IgG2a subclasses (data not shown). Sera from the Dsg3-immunized mice induced loss of adhesion of monolayers of human keratinocytes (Fig. 1B) to a greater extent than sera from mice transgenic for the unrelated HLA-DRB1*04:01 allele (Supplemental Fig. 2) whereas sera from PBS-injected HLA-DRB1*04:02–transgenic control mice did not (Fig. 1E). Injection of sera from the Dsg3-immunized HLA-DRB1*04:02–transgenic mice into human skin biopsies led to antiepithelial cell surface IgG deposits (Fig. 1C) and intraepidermal loss of adhesion, which is a hallmark of PV (Fig. 1D). In contrast, injection of sera from PBS-injected mice into human skin neither led to tissue-bound antiepithelial cell surface IgG (Fig. 1F) nor to intraepidermal loss of adhesion (Fig. 1G).

The in vivo–induced anti-human Dsg3–reactive IgG Abs recognized the same spectrum of epitopes as IgG autoantibodies from PV sera (20, 25). After the first immunization with human Dsg3, mouse sera preferentially reacted with the COOH-terminal Dsg3(EC5) subdomain and, after additional immunizations, also with the N-terminal Dsg3(EC1) and Dsg3(EC2) domains, which contain the major pathogenic B cell epitopes (Fig. 1H, 1I, Table I). Sera from the human Dsg3-immunized mice showed only little IgG cross-reactivity with mouse Dsg3, which shows an overall homology with human Dsg3 of 85.6% (Supplemental Fig. 3A). Despite the observed IgG reactivity of the mouse sera with several N- and COOH-terminal epitopes of the human Dsg3 ectodomain, there was no evidence for tissue-bound antiepithelial cell surface IgG Abs in oral mucosa of the Dsg3-immunized HLA-DRB1*04:02–transgenic mice, and accordingly, no evolving clinical phenotype (Supplemental Fig. 3B). Noteworthy, the amino acid sequences of the human and mouse Dsg3 ectodomain vary significantly in the areas of importance for CD4+ T cell activation (Table II), which may impede loss of T cell tolerance for mouse Dsg3, which precedes polyclonal activation of mouse Dsg3-specific pathogenic B cells. Comparison of the immundominant T cell epitopes of human Dsg3 with their mouse Dsg3 analogs revealed discordant amino acids at positions that are critical MHC class II anchor motifs. For example, the mouse analog to the human immunodominant T cell epitope of Dsg3, Dsg3(97–111), which is recognized by T cells from the majority of PV patients does not share critical HLA-DRB1*04:02–binding motifs at positions 4 and 6 (Table II).

HLA-DRB1*04:02–transgenic mice were treated with the anti-CD4 mAb GK 1.5 at the time of immunization with human Dsg3 leading to a complete inhibition of anti-Dsg3 IgG production (Fig. 2A). Furthermore, HLA-DRB1*04:02–transgenic mice were treated with the anti-CD40L mAb MR-1 before and right after immunization with human Dsg3 (Fig. 2B). Anti-Dsg3 IgG Ab production was completely inhibited, which demonstrates that T cell–B cell interaction is critical in the induction phase of Dsg3-specific IgG production. These findings in the HLA-DRB1*04:02–transgenic mouse model are in line with the clinical observation in three representative PV patients from a previously described cohort who were treated with the anti-CD20 mAb rituximab (26). Depletion of B cells by rituximab treatment (Fig. 2C) was associated with a rapid decrease of peripheral Dsg3-specific Th2 cells (Fig. 2D), followed by a more delayed decrease of anti-Dsg3 serum IgG (Fig. 2E).

Induction of human Dsg3-specific IgG Abs requires interaction between CD4+ T cells and B cells. (A) Treatment of the HLA-DRB1*04:02–transgenic mice with the anti-CD4 mAb GK1.5 at the time of immunization with human Dsg3 completely abrogates the formation of anti-Dsg3 IgG (n = 2). (B) In addition, treatment of the HLA-DRB1*04:02–transgenic mice with the anti-CD40L Ab MR-1 at the time of immunization and immediately afterward completely inhibits anti-Dsg3 IgG Ab induction (n = 2). (C) In three PV patients, treatment with the anti-CD20 Ab rituximab does not only (C) rapidly deplete peripheral B cells but also induces (D) a rapid decrease of peripheral IL-4–secreting Dsg3-reactive Th2 cells, (E) followed by a significant reduction of anti-Dsg3 serum IgG [(C–E) referring to Ref. 26].

Immunization of the HLA-DRB1*04:02-transgenic mice with human Dsg3 induced a T cell response against a set of T cell epitopes of Dsg3, which all bind to HLA-DRB1*04:02 (Fig. 3, Table III). Splenic CD4+ T cells from mice injected with human Dsg3 showed a proliferative response against a set of HLA-DRB1*04:02–binding Dsg3 peptides presented by BMDC from HLA-DRB1*04:02–transgenic mice (Table III) as shown by CSFE staining (Fig. 3A). In contrast, ex vivo challenge of the splenic CD4+ T cells with a set of Dsg3 peptides that do not bind to HLA-DRB1*04:02 (Table III) did not induce a significant proliferative response (Fig. 3B). Controls include cocultures of the splenic CD4+ T cells with BMDC alone (Fig. 3C) and BMDC with anti-CD3 mAb (Fig. 3D), respectively.

CD4+ T cells from human Dsg3-immunized HLA-DRB1*04:02–transgenic mice recognize a limited set of Dsg3 peptides. (A) Splenic CD4+ T cells from Dsg3-immunized HLA-DRB1*04:02–transgenic mice recognize a set of five Dsg3 peptides that bind to HLA-DRB1*04:02 (Table III) upon coculture with HLA-DRB1*04:02+ BMDC as shown by CSFE staining. (B) In contrast, CD4+ splenic T cells from Dsg3-immunized HLA-DRB1*04:02–transgenic mice show background proliferation in coculture with a set of Dsg3 peptides that do not bind to DRB1*04:02 (Table III). CD4+ splenic T cells in coculture with BMDC alone (C) and in coculture with BMDC and the anti-CD3 Ab 145-2c11 (D) [(A–D) representative results of two experiments]. (E and F) Draining lymph node cells from HLA-DR04:02–transgenic mice that were immunized with the five HLA-DRB1*04:02–binding Dsg3 peptides show IL-4+ and IFN-γ+ T cell responses upon in vitro stimulation with human Dsg3 or HLA-DRB1*04:02–binding Dsg3 peptides, respectively, but not with Dsg3 peptides that do not bind HLA-DRB1*04:02. Mice immunized with HLA-DRB1*04:02–nonbinding peptides did not show an IL-4+ or IFN-γ+ T cell response to Dsg3 [(E and F) n = 6–10]. (G and H) Lymph node cells from HLA-DRB1*04:01–transgenic mice immunized with HLA-DRB1*04:02–binding and –nonbinding Dsg3-peptides, respectively, showed both IL-4+ and IFN-γ+ T cell responses to the respective set of Dsg3 peptides, but there were neither IL-4+ (except for one mouse) nor IFN-γ+ T cell responses upon in vitro restimulation with Dsg3 [(G and H) n = 2–4].

In the reverse experiment, draining lymph node cells from HLA-DR04:02–transgenic mice, which had been immunized with a set of five HLA-DRB1*04:02-binding Dsg3 peptides (Table III, Supplemental Fig. 1C), showed both IL-4+ and IFN-γ+ T cell responses upon in vitro restimulation with human Dsg3 or HLA-DRB1*04:02–binding Dsg3 peptides, respectively, but not or only to a much lesser extent with Dsg3 peptides that do not bind HLA-DRB1*04:02 (Fig. 3E, 3F). HLA-DRB1*04:02–transgenic mice immunized with HLA-DRB1*04:02–nonbinding Dsg3 peptides showed merely an IFN-γ+ T cell response to the same set of peptides but neither IL-4+ nor IFN-γ+ T cell responses to Dsg3 (Fig. 3E, 3F). In contrast, splenic CD4+ T cells from HLA-DRB1*04:01–transgenic mice that had been immunized with the same set of HLA-DRB1*04:02–binding Dsg3 peptides (Table III) did neither show IL-4+ nor IFN-γ+ T cell responses against human Dsg3 in vitro (Fig. 3G, 3H). Immunization and in vitro restimulation with HLA-DRB1*04:02–binding Dsg3 peptides induced only an IL-4+ T cell response in a single HLA-DRB1*04:02–transgenic mouse, whereas immunization followed by in vitro challenge with HLA-DRB1*04:02–nonbinding Dsg3 peptides led to an IL-4+ and IFN-γ+ T cell response (Fig. 3G, 3H). The background stimulation of IL-4+ and IFN-γ+ lymph node T cells from HLA-DRB1*04:02–transgenic mice that were injected with PBS and adjuvant only is shown in Supplemental Fig. 4A and 4B.

Immunization of HLA-DRB1*04:02–transgenic mice with T cell epitopes of human Dsg3 leads to the induction of anti-Dsg3 IgG

Immunization of the HLA-DRB1*04:02–transgenic mice with a pool of HLA-DRB1*04:02–binding Dsg3 peptides (Table III) induced IgG Abs against human Dsg3 as shown by ELISA (Fig. 4A) and indirect immunofluorescence microscopy on monkey esophagus epithelium (Fig. 4B). In contrast, immunization of the HLA-DRB1*04:02–transgenic mice with Dsg3 peptides, which do not bind to HLA-DRB1*04:02 (Table III), did neither induce an IgG response against human Dsg3 by ELISA (Fig. 4A) nor did the sera react with monkey esophagus epithelium (Fig. 4C). Most strikingly, immunization of mice transgenic for HLA-DRB1*0401 (which is not related to PV and expresses different peptide binding motifs) with the HLA-DRB1*04:02–binding Dsg3 peptides did not induce IgG against Dsg3 as determined by ELISA (Fig. 4D) and indirect immunofluorescence on monkey esophagus (Fig. 4E). Neither did immunization of the HLA-DRB1*0401–transgenic mice with Dsg3 peptides that do not bind HLA-DRB1*04:02 (Table III) induce anti-Dsg3 IgG as determined by ELISA (Fig. 4D) and indirect immunofluorescence (Fig. 4F). These findings demonstrate that T cell recognition of HLA-DRB1*04:02–restricted epitopes of Dsg3 is critical for the induction of IgG Abs against Dsg3.

Immunization of HLA-DRB1*04:02–transgenic mice with HLA-DRB1*04:02–binding Dsg3 T cell epitopes induces IgG Abs against human Dsg3. (A) Immunization of HLA-DRB1*04:02–transgenic mice with a set of five HLA-DRB1*04:02–binding Dsg3 peptides (Table III) induces IgG against human Dsg3. In contrast, immunization of the mice with Dsg3 peptides that do not bind to DRB1*04:02 (Table III) does not induce anti-human Dsg3 IgG as shown by ELISA. Controls include IgG reactivity against Dsg3 of mice immunized with human recombinant Dsg3 (n = 3). (B) Anti-Dsg3 IgG-reactive sera from mice immunized with the HLA-DRB1*04:02–binding Dsg3 peptides stain the surface of epithelial cells of monkey esophagus (C) whereas sera from mice immunized with Dsg3 peptides that do not bind to HLADRB1*04:02 do not (B and C, representative results of three mice). In contrast, sera from HLA-DRB1*04:01–transgenic mice immunized with the HLA-DRB1*04:02–binding Dsg3 peptides or with the HLA-DRB1*04:02–nonbinding Dsg3 peptides, respectively, do not show anti-Dsg3 serum IgG reactivity (D) nor do they react with epithelial cells of monkey esophagus (E and F). (D) Controls include serum IgG reactivity against human Dsg3 of the HLA-DRB1*04:01–transgenic mice immunized with human Dsg3 [(D–F) n = 2–4]. (Serum samples were diluted 1:20 for ELISA analysis and 1:25 for immunofluorescence staining.)

Discussion

In this study, we show in an HLA-DRB1*04:02–transgenic mouse model of the autoimmune bullous skin disorder PV that T cell recognition of epitopes of Dsg3, the autoantigen of PV, in association with HLA-DRB1*04:02, leads, via B cell help, to the formation of pathogenic IgG Abs that induce loss of epidermal keratinocyte dissociation, a key finding in PV. On the basis of these observations, PV, although pathogenetically linked to IgG autoantibodies against the desmosomal cadherin, Dsg3 should be considered as a T cell–dependent autoimmune disorder and may largely profit from a therapeutic downregulation of autoaggressive T cells.

The hypothesis that autoreactive CD4+ T cells are critical initiators and perpetuators of the autoimmune pathology of PV is based on epidemiological observations which show a strong association of PV with two distinct HLA class II alleles, DRB1*04:02 and HLA-DQB1*05:03 (4, 5, 27, 28). HLA-DRB1*04:02 possesses a negative charge at positions DRβ70 (aspartate) and DRβ71 (glutamate) contributing to the shape and the charge of the p4 pocket which is critical for the binding of Dsg3 peptides for presentation to autoaggressive T cells in PV (6, 10). Both, DRB1*04:02 and HLA-DQB1*05:03, show a great homology in binding epitopes of the Dsg3 ectodomain, which is supported by previous findings of our group and others that both HLA class II alleles restrict T cell recognition of identical Dsg3 epitopes (Table III) (9, 29).

Previous studies from our group identified T cell responses to Dsg3 not only in PV patients but also in healthy carriers of the aforementioned PV-associated HLA class II alleles whose activation was found to be restricted by HLA-DRB1*04:02 and HLA-DQB1*05:03, respectively (8, 19, 30). In PV, Dsg3-specific autoaggressive T cells were predominantly of the Th2 type, whereas Dsg-reactive T cells in the healthy individuals were mainly Th1 cells (7–9). A similar dichotomy of autoreactive T cells in patients and healthy donors was also found in the pathogenetically related but distinct autoimmune bullous skin disorders, pemphigus foliaceus and bullous pemphigoid, which are also mediated by pathogenic IgG autoantibodies (31, 32).

Overall, PV can be considered as a Th2-driven autoimmune disorder because most of the autoantibodies belong to the IgG4 and IgE subclasses (7, 33–37). Moreover, patients with PV have significantly lower frequencies of peripheral IL-10–secreting Dsg3-reactive type 1 regulatory T (Tr1) cells than healthy carriers of HLA-DRB1*04:02 that suppress proliferative responses of T effector cells in vitro (38). Thus, activation of Dsg3-reactive effector T cells may be controlled by Dsg3-specific Tr1 cells leading to peripheral tolerance in healthy individuals who are protected by the higher frequency of Dsg3-reactive Tr1 cells than patients (38, 39).

Immunization of humanized HLA-DRB1*04:02–transgenic mice with T cell epitopes of Dsg3 is sufficient to induce a robust CD4+ T and B cell response against human Dsg3 leading to the production of pathogenic IgG Abs, which induced loss of adhesion of human epidermal keratinocytes ex vivo and in vitro (Fig. 4A, 4B). These IgG Abs only showed little cross-reactivity with mouse Dsg3, which exhibits an overall homology to human Dsg3 of 85.6% (40). Specifically, there is a higher degree of conservation (86–89% identity) for the N-terminal EC1 and EC2 ectodomains of Dsg3, which contain the major pathogenic autoantibody epitopes in PV patients (20, 25, 40). In contrast, the homology between the human and mouse COOH-terminal Dsg3(EC5) ectodomains is much lower (56% identity) (40). Still, the in vivo–induced anti-human Dsg3-specific IgG Abs did not sufficiently bind to the relevant mouse Dsg3 epitopes to induce a clinical phenotype in our HLA-transgenic animals. Moreover, the amino acid sequences of human and mouse Dsg3 vary significantly in the areas of importance for CD4+ T cell activation (Table II), which may impede loss of T cell tolerance for mouse Dsg3, which precedes polyclonal activation of mouse Dsg3-specific pathogenic B cells. In this respect, the present mouse model differs from the human autoimmune disease PV because immunization with human Dsg3 protein elicits an immune response to a foreign Ag in these animals. Thus, we are limited in analyzing loss of tolerance to endogenous mouse Dsg3 on both the CD4+ T cell and the B cell level. The current HLA-transgenic mouse model is suitable for investigating the effector phase of the autoimmune response in PV with particular emphasis on the activation and interaction of T and B cells, specific for human Dsg3, respectively. However, the primary scope of this investigation was to characterize cellular and humoral immune responses to human Dsg3 under in vivo, with emphasis on Dsg3 peptide recognition in association with HLA-B1*04:02, which is highly prevalent in PV.

The fine specificity of the HLA-DRB1*04:02–restricted CD4+ T cell response to Dsg3 is documented by the observation that immunization of the HLA-DRB1*04:02–transgenic mice with Dsg3 peptides that do not bind HLA-DRB1*04:02 neither induces Dsg3-reactive T cell responses nor Dsg3-specific IgG Abs (Figs. 3E, 3F, 4A–C). Reversely, immunization of mice transgenic for the unrelated HLA-DRB1*04:01 allele with HLA-DRB1*04:02–binding T cell epitopes of human Dsg3 does not lead to the formation of Dsg3-specific IgG (Fig. 4D–F). HLA-DRB1*04:01, which is associated with rheumatoid arthritis, differs from DRB1*04:02 only by the positive charge of the p4 pocket, which is critical for the binding of antigenic peptides (6, 41).

Immunization of HLA-DRB1*04:01 control mice with Dsg3 protein induced Dsg3-specific IgG as measured by Dsg3 ELISA (Fig. 4D). In the functional keratinocyte dissociation assay, however, the pathogenicity of anti-Dsg3 of the HLA-DRB1*04:01 mice tended to be lower compared with serum samples of the Dsg3-immunized HLA-DRB1*04:02–transgenic mice (Supplemental Fig. 2B). Of note, we did not see principal differences in the epitope specificity of the Dsg3-reactive IgG Abs in HLA-DRB1*04:01– and DR04:02–transgenic mice, respectively (data not shown). These findings suggest that the human Dsg3 protein also contains CD4+ T cell epitopes that bind to HLA-DR04:01 and, via T cell activation and T cell–B cell interaction, induce an IgG response against the human Dsg3 Ag.

Wucherpfennig et al. (6) proposed several candidate T cell peptides of Dsg3 on the basis of an algorithm for anchor motifs of the Dsg3 peptides and the charge of critical peptide binding pockets of DRB1*04:02. Moreover, they showed a proliferative in vitro response of peripheral lymphocytes from PV patients to three of the identified HLA-DRB1*04:02–associated peptides residing within the Dsg3 ectodomain (6). An independent study confirmed these findings and identified 10 HLA-DRB1*04:02–binding epitopes of Dsg3, which included the ones proposed previously by Wucherpfennig et al. based on molecular models (28). Using long-term CD4+ T cell clones, our group demonstrated that the majority of HLA-DRB1*04:02–positive PV patients showed a proliferative T cell response to these HLA-DRB1*04:02–binding Dsg3 T cell peptides (Table III) (8, 9, 42). Specifically, all of the identified Dsg3 epitopes share common anchor residues at relative positions 1, 4, and 6, which were previously identified to be potential anchor motifs for DRB1*04:02 and carry a positive charge at position 4, which is critical for binding to the negatively charged P4 pocket of DRB1*04:02 (Table III) (6, 42).

In this study, we show that induction of anti-human Dsg3 IgG Abs in immunized HLA-DRB1*04:02–transgenic mice depends on the interaction of CD4+ T cells and B cells (Fig. 2). This in vivo finding closes an important gap that was not yet fully proven in the human disorder PV. Circumstantial evidence for a pathogenetically relevant interaction of autoreactive CD4+ T and B cells came from the clinical observation that in PV patients therapeutic B cell depletion with the monoclonal anti-CD20 mAb rituximab leads to a downregulation of Dsg3-specific T cells (Fig. 2C, 2D), which is associated with a prompt clinical improvement before anti-Dsg3 serum IgG autoantibodies are decreased (Fig. 2E) (26). Noteworthy, the frequencies of tetanus toxoid (TT)–specific Th cells and serum IgG were not affected by rituximab treatment (26). This observation strongly suggests that Dsg3-reactive T cells largely depend on B cells as APCs whereas TT-specific T cells do not. This finding is in line with previous studies in rheumatoid arthritis which showed that serum IgG titers against pathogens such as pneumococcal capsular polysaccharides or TT were not significantly affected by rituximab (43). The dramatic inhibitory effect of rituximab on anti-Dsg3 IgG serum levels strongly suggests that their secretion largely depends on short-lived autoreactive plasma cells (44). Moreover, Dsg3-specific IgG-producing B cells were detected in PV patients by ELISPOT assay upon ex vivo stimulation of their peripheral lymphocytes with Dsg3 (45). When the patients’ peripheral lymphocytes were depleted of CD4+ T cells, anti-Dsg3 IgG–producing B cells were no longer detectable (45).

The critical role of T cell–B cell interaction in the PV pathogenesis is also supported by a PV mouse model established by Amagai and coworkers (46, 47). Transfer of splenocytes of Dsg3−/− mice immunized with murine Dsg3 into Dsg3+/+Rag2−/− recipient mice led to a clinical phenotype with mucosal erosions reminiscent of PV. In contrast, transfer of splenocytes depleted of CD4+ T cells to the Dsg3+/+Rag2−/− mice did neither induce autoantibody production nor a PV-like phenotype. Neither did the transfer of B cell–depleted splenocytes into Dsg3+/+Rag2−/− mice lead to the induction of anti-Dsg3 IgG and/or a clinical phenotype. Moreover, using the same animal model, Takahashi et al. (48) showed that a single Dsg3-reactive T cell clone was sufficient to prime naive B cells to produce Dsg3-specific pathogenic IgG autoantibodies. In an independent mouse model, immunization of mice with human Dsg3 led to the induction of Dsg3-reactive Th2 cells that were able to render unprimed B cells to secrete anti-Dsg3 IgG (49).

In summary, the current study demonstrates that CD4+ T cell recognition of Dsg3, the autoantigen of PV is tighly regulated by HLA-DRB1*04:02, which is prevalent in PV. Using DRB1*04:02–transgenic mice, we show that T cell–dependent B cell activation is critical for the induction of pathogenic IgG Abs, which directly induce epidermal loss of adhesion, a key finding in the immune pathogenesis of PV. Thus, specific targeting of Dsg3-reactive CD4+ T cells holds major promise as a therapeutic option in PV. A similar approach has proven successful in the therapeutic downregulation of autoaggressive T cell responses in patients with multiple sclerosis (50, 51).

Disclosures

R.E. has consultant arrangements with and has received payments for lectures from Novartis and received grants and payments for lectures and travel expenses from Fresenius Medical Care; M.H. has board memberships with Novartis and GlaxoSmithKline Stiefel, has consultant arrangements with Roche, Biogen Idec, and Union Chimique Belge, received payments for lectures from Janssen-Cilag and Biogen Idec, and received travel support from Janssen-Cilag; and J.W. is now employed by Miltenyi Biotec. The other authors have no financial conflicts of interest.

Acknowledgments

We thank Eva Podstawa and Susanne Schwietzke for excellent technical assistance and Drs. Ralf Müller, Angela Nagel, and Christian Möbs for scientific advice and discussions during the evolution of the project. We also thank Dr. Roland Martin for thoughtful discussion and critical comments on the manuscript.

Footnotes

This work was supported by Grants EM 80/1-1 (to R.E.) and HE 1602/7-2 (to R.E. and M.H.) from the German Research Foundation (Deutsche Forschungsgemeinschaft); grants from the Knut and Alice Wallenberg Foundation (to R.H.), the Swedish Medical Research Council, and the Swedish Foundation for Strategic Research; and European Union Grants MASTERSWITCH (HEALTH-F2-2008-223404), BeTheCure (IMI115142) (to R.H. and J.B.), and HEALTH-F2-2008-200515 “Pemphigus – from Autoimmunity to Disease” (to M.H.). M.H. received grants from Fresenius Medical Care and Medical & Biological Laboratories, Co., Ltd. G.S. received grants from the National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases and the Juvenile Diabetes Research Foundation.

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